Desulfurization compound bacterium with high desulfurization characteristic and application thereof

Abstract

The invention discloses a desulfurization compound bacterium with a high desulfurization characteristic and an application of the desulfurization compound bacterium. The desulfurization compound bacterium disclosed by the invention is prepared from Pristeria megaterium A-1 and Exiguobacterium aceticum C-1, and the desulfurization compound bacterium is prepared from the following raw materials: the P.megaterium A-1 and the Exiguobacterium aceticum C-1, the Exiguobacterium aceticum C-1, the Exiguobacterium aceticum C-1 and the Exiguobacterium aceticum C-1 and the Exiguobacterium aceticum C-1, the desulfurization compound bacterium has the characteristic of high desulfurization, can be used for treating wastewater (such as freshwater aquaculture wastewater) with the sulfide content of 40-150mg / L, provides a healthy growth environment for aquatic animals, and improves the aquaculture benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and wastewater treatment, and specifically relates to a desulfurization composite bacterium with high desulfurization performance and application thereof. Background Art

[0002] With the rapid development of my country's aquatic industry, the environmental problems of high-density and intensive aquaculture have become increasingly prominent. Excessive fertilization leads to excess nutrients in the water body, and the enrichment of nutrients will cause a series of environmental and ecological problems. On the one hand, excessive feeding leads to the loss of bait, which directly causes economic losses. On the other hand, the rotting and decomposition of bait deposited at the bottom of the aquaculture pond will consume a lot of oxygen and deteriorate the local environment of the bottom. In short, a large amount of leftover bait, dead animal and plant bodies, excrement, and fertilizers are deposited in the pond mud, which is very likely to produce harmful substances such as ammonia, organic acids, sulfides, and methane, which endanger aquaculture production.

[0003] Sulfide is a harmful product of high-density, large-scale feeding aquaculture methods that damage the aquaculture ecological environment and weaken the self-purification ability of water bodies. Bottom sulfide is one of the important factors affecting the aquaculture environment and an important indicator to measure the quality of the bottom environment. Moreover, hydrogen sulfide itself, as a strong reducing agent, can affect the normal metabolism of most aerobic microorganisms and algae, thereby weakening the self-purification ability of water bodies. In addition, sulfide can combine with hemoglobin in the blood of farmed organisms to produce sulfhemoglobin, generating chocolate-like black blood, which reduces the oxygen-carrying capacity of the body's blood. Sulfide has a strong irritating and corrosive effect on the gill tissue of farmed organisms, causing tissue coagulative necrosis, resulting in difficulty breathing in the organism and even suffocation and death. The toxicity of sulfide to fish and shrimp is mainly through the mucous membrane on the gill surface and the Na in the tissue + or Cu in blood 2+ They combine to form substances with strong irritating effects, inhibit the occurrence of certain enzymatic reactions, and cause harm to the body.

[0004] At present, there are few studies on the screening of freshwater desulfurization bacteria. Therefore, isolating and screening desulfurization bacteria with high desulfurization rates is of great significance for aquaculture wastewater treatment, maintaining a healthy aquaculture environment, and improving aquaculture benefits. Summary of the invention

[0005] In order to better treat aquaculture wastewater, maintain a healthy growth environment for aquatic animals, and improve aquaculture benefits, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides a desulfurization composite bacterium with high desulfurization characteristics, wherein the desulfurization composite bacterium is composed of Priestia megaterium A-1 and Exiguobacterium acetylicum C-1.

[0007] The Priestia megaterium A-1 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO.33038 and the deposit date December 12, 2024.

[0008] The Exiguobacterium acetylicum C-1 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO.33039 and the deposit date December 12, 2024.

[0009] Preferably, the 16S rDNA sequence of the Priesteria megaterium A-1 is as shown in SEQ ID NO: 1, and the specific sequence is as follows:

[0010]

[0011] Preferably, the 16S rDNA sequence of Exiguobacterium acetylicum C-1 is as shown in SEQ ID NO:2.

[0012] The specific sequence is as follows:

[0013]

[0014] Preferably, in the desulfurization composite bacteria, the effective live bacteria count ratio of the Prioccludinium giganteum A-1 to the Exiguobacterium acetylicum C-1 is 5:1 to 1:5, for example: 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5.

[0015] Furthermore, the effective viable bacterial count ratio of the Prioccidioides megaterium A-1 to the Exiguobacterium acetylicum C-1 is 1:3.

[0016] In a second aspect, the present invention provides a method for culturing the desulfurization composite bacteria described in the first aspect, the culturing method comprising using a culture medium, the concentration of sulfide in the culture medium being 1.0 to 3.0 mg / mL, for example: 1.0 mg / mL, 1.4 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2.0 mg / mL, 2.2 mg / mL, 2.4 mg / mL, 2.6 mg / mL, 2.8 mg / mL, 3.0 mg / mL.

[0017] Preferably, the basic formula of the culture medium is as follows:

[0018] Glucose 5.0g, KH 2 PO 4 1.0 g, K 2 HPO 4 1.0 g, magnesium chloride hexahydrate 0.8 g, FeCl 2 0.01g, NH 4 Cl0.4g, NaHS 5.0g, distilled water 1000mL, pH value is 6.8-7.2.

[0019] Preferably, the culture method further comprises inoculating the desulfurization composite bacteria into synthetic wastewater.

[0020] Furthermore, the basic formula of the synthetic wastewater is: 3.0 g glucose, KH 2 PO 4 1.0 g, Na 2 S3.0g, distilled water 1000mL, pH value 6.8-7.2.

[0021] In a third aspect, the present invention provides a bacterial agent, wherein the bacterial agent comprises the desulfurization composite bacteria described in the first aspect.

[0022] Preferably, the effective viable count of Priesteria gigantea A-1 in the bacterial agent is 1×10 8 ~9×10 9 CFU / mL, for example: 1×10 8 CFU / mL, 3×10 8CFU / mL, 6×10 8 CFU / mL, 8×10 8 CFU / mL, 1×10 9 CFU / mL, 3×10 9 CFU / mL, 6×10 9 CFU / mL, 8×10 9 CFU / mL, 9×10 9 CFU / mL.

[0023] Alternatively, the OD of Priesteria gigantea A-1 in the bacterial agent is 600 =0.6~1.0, for example: 0.6, 0.7, 0.8, 0.9, 1.0.

[0024] Preferably, the effective viable count of Exiguobacterium acetylicum C-1 in the bacterial agent is 1×10 8 ~9×10 9 CFU / mL, for example: 1×10 8 CFU / mL, 3×10 8 CFU / mL, 6×10 8 CFU / mL, 8×10 8 CFU / mL, 1×10 9 CFU / mL, 3×10 9 CFU / mL, 6×10 9 CFU / mL, 8×10 9 CFU / mL, 9×10 9 CFU / mL.

[0025] Alternatively, the OD of Exiguobacterium acetylicum C-1 in the bacterial agent is 600 =0.6~1.0, for example: 0.6, 0.7, 0.8, 0.9, 1.0.

[0026] Preferably, the bacterial agent may also include non-oxygen-producing photosynthetic bacteria such as Rhodopseudomonas (such as Rhodopseudomonas palustris) and green non-sulfur bacteria.

[0027] In a fourth aspect, the present invention provides a wastewater treatment agent, which comprises the desulfurization composite bacteria described in the first aspect or the bacterial agent described in the third aspect.

[0028] Preferably, the wastewater treatment agent may also include agents for purifying water quality, such as polyaluminium chloride, polyacrylamide, activated carbon or diatomaceous earth.

[0029] In a fifth aspect, the present invention provides use of the desulfurization composite bacteria described in the first aspect or the bacterial agent described in the third aspect in the preparation of a wastewater treatment agent.

[0030] In a sixth aspect, the present invention provides use of the desulfurization composite bacteria described in the first aspect, the bacterial agent described in the third aspect, or the wastewater treatment agent described in the fourth aspect in treating wastewater.

[0031] Preferably, the sulfide concentration in the wastewater is ≥40 mg / L.

[0032] Furthermore, the sulfide concentration in the wastewater is ≤150 mg / L, for example: 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, 100 mg / L, 110 mg / L, 120 mg / L, 130 mg / L, 140 mg / L, 150 mg / L.

[0033] Furthermore, the sulfide includes but is not limited to H 2 S, HS - , S 2- , soluble sulfides and acid-soluble metal sulfides.

[0034] Further, the sulfide comprises H 2 S, HS - , S 2- Any one or a combination of two or more.

[0035] Preferably, the desulfurization composite bacteria, the bacterial agent or the wastewater treatment agent is added to the wastewater to remove the sulfide.

[0036] Preferably, the wastewater is aquaculture wastewater, more preferably freshwater aquaculture wastewater.

[0037] Preferably, the inoculation amount of the desulfurization composite bacteria is 1-5%, for example: 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%.

[0038] The terms “include” or “comprising” described in the present invention are open-ended expressions, which include the specified components or steps described, and other specified components or steps that will not be substantially affected.

[0039] The term "and / or" of the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A", "A and B" and "B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0040] Beneficial effects of the present invention:

[0041] The desulfurization composite bacteria of the present invention are composed of Priestia megaterium A-1 and Exiguobacterium acetylicum C-1, have high desulfurization characteristics, can be used to treat wastewater with a sulfide content of 40 to 150 mg / L (such as freshwater aquaculture wastewater), provide a healthy growth environment for aquatic animals, and improve the efficiency of aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The colony morphology and Gram staining images of the strains, A is Priesteria gigantea A-1, B is Exiguobacterium acetylicum C-1;

[0043] Figure 2 The figure shows the effect of different bacterial solution ratios on the desulfurization performance of composite bacteria;

[0044] Figure 3 The effect of different C / S ratios on the desulfurization performance of composite bacteria;

[0045] Figure 4 The effect of different pH on the desulfurization performance of composite bacteria;

[0046] Figure 5 The effect of different temperatures on the desulfurization performance of composite bacteria;

[0047] Figure 6 This is a comparative growth curve of sulfide degradation by composite bacteria and single strain.

[0048] Culture deposits for patent procedures:

[0049] Priestia megaterium A-1, the deposit number is CGMCC NO.33038, and the deposit date is December 12, 2024.

[0050] Exiguobacterium acetylicum C-1, the preservation number is CGMCC NO.33039, and the preservation date is December 12, 2024.

[0051] Depository: China General Microbiology Center (CGMCC).

[0052] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Postal Code: 100101. DETAILED DESCRIPTION

[0053] The technical scheme of the present invention is described clearly and completely below in conjunction with the embodiments and drawings. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0054] The culture medium involved in the present invention is:

[0055] Enrichment medium: glucose 5.0g, KH 2 PO 4 1.0 g, K 2 HPO 4 1.0 g, magnesium chloride hexahydrate 0.8 g, FeCl 2 0.01 g, NH 4 Cl 0.4g, NaHS 5.0g, distilled water 1000mL, pH value 6.8-7.2, sterilize at 115℃ for 30min.

[0056] Separation solid medium: glucose 5.0g, KH 2 PO 4 1.0 g, K 2 HPO 4 1.0 g, magnesium chloride hexahydrate 0.8 g, FeCl 2 0.01 g, NH 4 Cl 0.4g, NaHS 5.0g, distilled water 1000mL, agar powder 17g, pH value 6.8-7.2, sterilize at 115℃ for 30min.

[0057] Synthetic wastewater: glucose 3.0g, KH 2 PO 4 1.0 g, Na 2 S 3.0g, distilled water 1000mL, pH value is 6.8-7.2; due to the volatilization of sulfide during the preparation process, the sulfide concentration is 90-150mg / L.

[0058] LB medium: 10 g tryptone, 5 g yeast powder, 10 g sodium chloride, 1 L distilled water, pH 7.0, sterilized at 121°C for 20 min.

[0059] Example 1 Isolation, screening and identification of desulfurization strains with high desulfurization rates

[0060] Take 10g of Benxi Guanyin Pavilion sediment sample and place it in 90mL enrichment culture medium, place it in a shaker at 30℃ and 180rpm for constant temperature culture for 1 day, take 10mL of enriched bacterial solution and culture it in 90mL enrichment culture medium, repeat this three times; then take the bacterial solution for gradient dilution and spread it on the surface of separation solid culture medium, after constant temperature culture at 30℃ for 1 to 3 days, select different strains with good growth and further purify them on the separation solid culture medium, pick single colonies for streaking separation, repeat this three times to obtain a single strain; pick a single colony with an inoculation loop and add it to synthetic wastewater, use methylene blue spectrophotometry to measure the strain with the strongest desulfurization ability, and store this single strain in a -80℃ refrigerator.

[0061] The morphological identification results are as follows Figure 1 As shown, the surface of the screened colonies is moist, smooth, opaque, with complete and regular edges, presenting round protrusions, and the colonies are light yellow.

[0062] Molecular biological identification: The DNA of the screened strains was extracted using a bacterial genome extraction kit (Tiangen), and the conserved target fragments were PCR amplified using the 16S rDNA gene universal primers 27F (SEQ ID NO: 3) and 1492R (SEQ ID NO: 4), and the amplified products were cut and recovered, followed by sequencing analysis. PCR amplification, sequencing and BLAST comparison. The results showed that among the 16S rDNA gene sequences of the two strains screened, one strain with a sequence as shown in SEQ ID NO: 1 had a similarity of 99.99% with Priestia megaterium. Another strain with a sequence as shown in SEQ ID NO: 2 had a similarity of 99.99% with Exiguobacterium acetylicum. Therefore, strain A-1 was determined to be Priestia megaterium, named Priestia megaterium A-1; strain C-1 was Exiguobacterium acetylicum, named Exiguobacterium acetylicum C-1.

[0063] The specific sequence of 16S rDNA of Priesteria gigantea A-1 is as follows:

[0064]

[0065] The specific sequence of 16S rDNA of Exiguobacterium acetylicum C-1 is as follows:

[0066]

[0067] 27F:5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO:3)

[0068] 1492R:5'-GGTTACCTTGTTACGACTT-3'(SEQ ID NO:4)

[0069] Example 2 Effect of different bacterial liquid ratios on the desulfurization effect of composite bacteria

[0070] Seed solution preparation: Pick strains of Priesteria gigantea A-1 and Exiguobacterium acetylicum C-1 and place them in LB medium, shake and culture at 30°C and 180 rpm for 10-12 h, and take the culture medium to measure the OD of the solution. 600 OD 600 When the value reaches 0.6-0.8, it indicates that the strain has entered the logarithmic growth phase and the seed solution can be used normally. The viable counts of Priesteria gigantea A-1 and Exiguobacterium acetyl C-1 are both 6-8×10 8 CFU / mL.

[0071] The bacterial solution ratios of Priesteria gigantea A-1 and Exiguobacterium acetyl C-1 were adjusted to A-1:C-1=5:1, A-1:C-1=3:1, A-1:C-1=1:1, A-1:C-1=1:3, and A-1:C-1=1:5, respectively. The composite seed solution of each ratio was inoculated into 100 mL of synthetic wastewater at a 3.0% inoculation amount, pH=7, 25°C, and 180rpm shaking culture for 24 hours. After 24 hours, water samples were taken to measure the sulfide content in the solution by methylene blue spectrophotometry. The initial synthetic wastewater with the same culture conditions and time was used as the blank control group, and the desulfurization rate was calculated according to the following formula.

[0072] Desulfurization rate = (sulfide concentration of blank control group - sulfide concentration of water sample) / sulfide concentration of blank control group.

[0073] like Figure 2 As shown, the desulfurization rate of the composite bacteria was above 92% when A-1:C-1=5:1-1:5, and the highest desulfurization rate reached 95% when the ratio was 1:3.

[0074] Example 3 Effect of carbon-sulfur ratio (C / S) on desulfurization effect of composite bacteria

[0075] Seed solution preparation: same as Example 2.

[0076] The C / S of the synthetic wastewater was adjusted to 1:1, 3:1, 5:1, 7:1, and 9:1, respectively. The composite seed solution of A-1:C-1=1:3 was inoculated with 3.0% inoculation amount into 100 mL of synthetic wastewater with different C / S. The mixture was cultured at pH=7, 25°C, and 180rpm for 24 h. After 24 h, water samples were taken and the sulfide content was determined by methylene blue spectrophotometry, and the desulfurization rate was calculated (same as Example 2).

[0077] like Figure 3 As shown, when C / S is 1:1-9:1, the desulfurization rate of the composite bacteria is higher than 84%; when C / S is 1:1-7:1, the desulfurization rate of the composite bacteria is higher than 93%; when C / S is 1:1-3:1, the desulfurization rate is higher than 95%; when C / S is 3:1, the desulfurization rate reaches 98%.

[0078] Example 4 Effect of pH value on desulfurization effect of composite bacteria

[0079] Seed solution preparation: same as Example 2.

[0080] The pH values ​​of the synthetic wastewater were adjusted to 5.5, 6, 6.5, 7, 7.5, and 8, respectively. The composite seed solution of A-1:C-1=1:3 was inoculated with 3.0% inoculation amount into 100 mL of synthetic wastewater with different pH values. The culture conditions were C / S=3:1, 25°C, and 180 rpm shaking for 24 h. After 24 h, water samples were taken and the sulfide content was determined by methylene blue spectrophotometry, and the desulfurization rate was calculated (same as in Example 2).

[0081] like Figure 4 As shown, when the pH value is 6-8, the desulfurization rate of the composite bacteria for wastewater is higher than 85%; when the pH value is 6.5-7.5, the desulfurization rate is higher than 93%; when the pH value is 7.0, the desulfurization rate reaches 97%.

[0082] Example 5 Study on the effect of temperature on the desulfurization effect of composite bacteria

[0083] Seed solution preparation: same as Example 2.

[0084] The temperature of the synthetic wastewater was adjusted to 15°C, 20°C, 25°C, 30°C, and 35°C, respectively. The composite seed solution of A-1:C-1=1:3 was inoculated with 3.0% inoculation amount into 100 mL of synthetic wastewater at different temperatures. The pH was 7, C / S was 3:1, and the culture was shaken at 180 rpm for 24 h. After 24 h, water samples were taken and the sulfide content was determined by methylene blue spectrophotometry, and the desulfurization rate was calculated (same as Example 2).

[0085] like Figure 5 As shown, when the temperature is 15-35°C, the desulfurization rate of the composite bacteria for wastewater is higher than 90%; when the temperature is 20-30°C, the desulfurization rate is higher than 93%; at 20°C, the desulfurization rate reaches 97%.

[0086] Example 6 Evaluation of the desulfurization effect of composite bacteria on simulated wastewater

[0087] Seed solution preparation: same as Example 2.

[0088] The composite seed solution of A-1:C-1=1:3 was inoculated into 500 mL of synthetic wastewater at a 3.0% inoculation rate, pH=7, C / S=3:1, 20°C, and 180 rpm shaking culture for 24 h. Water samples were taken every 6 h to determine the sulfide concentration and solution OD 600 .

[0089] like Figure 6 As shown in the figure, in the range of 0 to 48 hours, compared with the blank control group, the sulfur content in the wastewater treated by Priesteria gigantea A-1, Exiguobacterium acetylicum C-1, and composite bacteria was lower than that in the blank control group, and the sulfur content was in the order of blank control group, Priesteria gigantea A-1, Exiguobacterium acetylicum C-1, and composite bacteria. In the range of 0 to 30 hours, in the order of high to low wastewater desulfurization rates, it was composite bacteria, Exiguobacterium acetylicum C-1, and Priesteria gigantea A-1. After 30 hours of wastewater treatment, the desulfurization rate of the composite bacteria was 87.31%, the desulfurization rate of Exiguobacterium acetylicum C-1 was 85.10%, and the desulfurization rate of Priesteria gigantea A-1 was 65.69%; after 36 hours of wastewater treatment, as the desulfurization rate gradually increased, the difference in desulfurization rate between Priesteria gigantea A-1, Exiguobacterium acetylicum C-1 and the composite bacteria gradually became smaller. At 36 hours, the desulfurization rate of the composite bacteria was 92.99%, the desulfurization rate of Exiguobacterium acetylicum C-1 was 92.99%, and the desulfurization rate of The desulfurization rate of the composite bacteria was 93.45%, and the desulfurization rate of Priesteria gigantea A-1 was 84.72%; at 42h, the desulfurization rate of the composite bacteria was 98.70%, the desulfurization rate of Exiguobacterium acetylicum C-1 was 98.13%, and the desulfurization rate of Priesteria gigantea A-1 was 96.21%; at 48h, the desulfurization rate of the composite bacteria was 99.21%, the desulfurization rate of Exiguobacterium acetylicum C-1 was 98.87%, and the desulfurization rate of Priesteria gigantea A-1 was 97.90%. Within 48h, the composite bacteria can remove more than 99% of the sulfide in the wastewater, and the desulfurization effect is better than that of Exiguobacterium acetylicum C-1 and Priesteria gigantea A-1.

[0090] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A desulfurization composite bacteria with high desulfurization performance, characterized in that: The desulfurization composite bacteria are composed of Priestia megaterium A-1 and Exiguobacterium acetylicum C-1; The Priestia megaterium A-1 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC NO.33038 and a deposit date of December 12, 2024; The Exiguobacterium acetylicum C-1 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO.33039 and the deposit date December 12, 2024.

2. The desulfurization composite bacteria according to claim 1, characterized in that: In the desulfurization composite bacteria, the effective live bacterial count ratio of the Prioccidioides megaterium A-1 to the Exiguobacterium acetylicum C-1 is 5:1 to 1:

5.

3. A bacterial agent, characterized in that: The bacterial agent comprises the desulfurization composite bacteria according to claim 1 or 2.

4. The bacterial agent according to claim 3, characterized in that The effective viable counts of Priesteria gigantea A-1 and Exiguobacterium acetylicum C-1 in the bacterial agent were both 1×10 8 ~9×10 9 CFU / mL.

5. A wastewater treatment agent, characterized in that: The wastewater treatment agent comprises the desulfurization composite bacteria described in any one of claims 1-2 or the bacterial agent described in any one of claims 3-4.

6. Use of the desulfurization composite bacteria according to any one of claims 1-2 or the bacterial agent according to any one of claims 3-4 in the preparation of a wastewater treatment agent.

7. Use of the desulfurization composite bacteria according to any one of claims 1-2, the bacterial agent according to any one of claims 3-4, or the wastewater treatment agent according to claim 5 in treating wastewater.

8. The use according to claim 7, characterized in that: The sulfide concentration in the wastewater is 40-150 mg / L.

9. The use according to claim 8, characterized in that: The sulfide includes H2S, HS - , S 2- Any one or a combination of two or more.

10. The use according to any one of claims 7 to 9, characterized in that: Adding the desulfurization composite bacteria, the bacterial agent or the wastewater treatment agent to the wastewater; Preferably, the inoculation amount of the desulfurization composite bacteria is 1-5%.

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